Pdgf as a biomarker for predicting resistance or effect of c-met targeting drugs

ABSTRACT

Provided is a method for evaluating efficacy of, or resistance to, a c-Met targeting agent including measuring a level of a PDGF protein and/or a PDGF coding gene.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of Korean Patent Application No.10-2014-0111704 on Aug. 26, 2014 with the Korean Intellectual PropertyOffice, the entire disclosure of which is hereby incorporated byreference.

INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY SUBMITTED

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted herewith and identifiedas follows: One 130,048 byte ASCII (Text) file named “719715_ST25.TXT,”created Aug. 26, 2015.

BACKGROUND

1. Field

Provided are a biomarker PDGF (platelet-derived growth factor) forevaluating efficacy of, or resistance to, a c-Met targeting agent and amethod for evaluating efficacy of, or resistance to, a c-Met targetingagent including measuring a level of a PDGF protein and/or a PDGF codinggene.

2. Description of the Related Art

A biomarker generally refers to a measured characteristic which may beused as an indicator of some change caused in an organism by an externalfactor. Recent studies have been made to apply biomarkers to thediagnosis of various diseases and the prediction or monitoring oftherapeutic effects of some agents. Among biomarkers relevant to drugdevelopment are pharmacodynamic markers (PD markers) for indicatingwhether drugs are functionally effective in vivo, and predictive markersfor indicating the most likely response to particular drugs beforeadministration. The use of such markers is helpful in establishing theclinical strategy of drugs. For example, a predictive marker, designedto indicate sensitivity or resistance to drug action, may be applied tothe selection of patients to allow for more effective drug therapy whilethe action mode of a drug in individual patients can be monitored with aPD marker, which together can lead to the establishment of effectivetherapeutic strategies. Further, even in the absence of a predictivemarker, a PD marker permits the early monitoring of responses to a drug,thus discriminating a drug-effective group from a drug-ineffective groupin an early stage. Consequentially, more effective and successful drugtherapies can be materialized. In addition, when applied to themonitoring of responses to a drug as a function of concentrations, a PDmarker can be an index for calculating suitable doses of the drug.

Meanwhile, a cancer is one of the leading causes of death. Although thedevelopment of medical techniques has brought about a remarkableprogress in cancer therapy, the five-year survival rate has onlyimproved by ten percent over the past two decades. This is becausecancer characteristics, such as rapid growth, metastasis, etc., make itdifficult to diagnose and treat within a suitable time. The introductionof suitable biomarkers to cancer therapy would identify thecharacteristics of cancer to increase the opportunity of applying asuitable therapeutic at an optimal time, whereby cancer treatment couldreach high success rates. For example, patients with lung cancer maydiffer from each other in cancer classification, genotype, and proteinsecretion, and thus must be treated with different, proper therapeutics.For chemotherapy using a specific drug, a corresponding biomarker, ifpresent, would reduce the number of erroneous trials and increasepossibility of success. In this regard, it is very important to explorebiomarkers for predicting or monitoring the effect of anti-cancertherapeutics. A proper biomarker, if successfully exploited, can make agreat contribution to the utility and value of anti-cancer drugs and thesuccess rate of treatment with them.

c-Met is a hepatocyte growth factor (HGF) receptor. HGF acts as amulti-functional cytokine which binds to the extracellular domain of thec-Met receptor to regulate cell division, cell motility, andmorphogenesis in various normal and tumor cells. The c-Met receptor is amembrane receptor that possesses tyrosine kinase activity. c-Met is aproto-oncogene, per se, that encodes the representative receptortyrosine kinase. Occasionally, it takes part in a variety of mechanismsresponsible for the development of cancer, such as oncogenesis, cancermetastasis, the migration and invasion of cancer cells, angiogenesis,etc., irrespectively of the ligand HGF, and thus has attracted intensiveattention as a target for anti-cancer therapy. Targeted therapies, suchas antibodies against c-Met, have been and are currently beingdeveloped.

In order to increase the effect of therapies using the developed c-Mettargeting drugs, it is important to develop biomarkers for predictingthe effect of the c-Met targeting drugs to select a subject who issuitable for application of the c-Met targeting drugs, and/or formonitoring the responsiveness of a patient who has been treated with thec-Met targeting drugs to establish more effective treatment strategiesusing the c-Met targeting drugs.

SUMMARY

Provided is a method for evaluating efficacy of a c-Met targeting agenton a patient with cancer, or the patient's resistance to the c-Mettargeting agent. The method comprises measuring PDGF protein level, mRNAexpression level of a gene encoding PDGF, or both, in a sample obtainedfrom a patient with cancer before administration of a c-Met targetingagent to the patient, and in a sample obtained from the patient afteradministration of a c-Met targeting agent to the patient; comparing thePDGF protein level of the samples, mRNA expression level of geneencoding PDGF, or both, of the samples obtained before administration ofthe c-Met targeting agent to that of the samples obtained afteradministration of the c-Met targeting agent, and evaluating whether thec-Met targeting agent is effective in treating the cancer based on achange in PDGF protein level, mRNA expression level of a gene encodingPDGF, or both. An increase in the PDGF protein level or the mRNAexpression level of gene encoding PDGF in the sample from the patientafter administration of the c-Met targeting agent, compared to the PDGFprotein level or the mRNA expression level in the sample beforeadministration of the c-Met targeting agent, indicates that the c-Mettargeting agent is not efficacious in treating the cancer or the patienthas resistance to the c-Met targeting agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the expression of various human growth factors in EBC1 andEBC1-SR#6 cell lines with acquired resistance to an anti-c-Met antibody.

FIG. 2 is a graph showing % of cell viability according to PDGF Receptor(PDGFR) inhibitor Ponatinib concentration, when EBC1 cell lines andEBC1-SR#6 cell lines were treated with Ponatinib and then cultured 72hours.

FIG. 3 is a graph showing % of cell viability according to FGFRinhibitor PD173074 concentration, when EBC1 and EBC1-SR#6 cell lineswere treated with PD173074 and then cultured 72 hours.

DETAILED DESCRIPTION

It is observed that the level of PDGF in a cell with acquired resistanceto a c-Met targeting agent by repeatedly treating the cell with thec-Met targeting agent, is significantly increased compared to that of acell with no resistance. In addition, when a resistance to a c-Mettargeting agent was induced, administrating an agent inhibiting PDGFsignal transduction pathway leads to an increase of chemosensitivity ofcancer cells. Therefore, efficacy of the c-Met targeting agent may beevaluated by measuring increase or decrease of PDGF level therebypredicting whether an innate resistance to a c-Met targeting agent(e.g., an anti-c-Met antibody) is present or an acquired resistance to ac-Met targeting agent (e.g., an anti-c-Met antibody) is induced byrepeated treatment with the c-Met targeting agent.

In addition, the measurement of the expression level of PDGF in abiological sample can provide information for predicting an efficacy of,or resistance to, a c-Met targeting agent on the biological sample or apatient from whom the biological sample is isolated. This informationmay be used for selecting a subject who is suitable for applying thec-Met targeting agent, or determining whether or not a c-Met targetingagent can achieve a desired anticancer effect. Based thereon, uses ofPDGF as a biomarker for predicting and/or evaluating an efficacy or aresistance of a c-Met targeting agent are provided.

In this disclosure, the efficacy of a c-Met targeting agent may refer toan effect of preventing, improving, alleviating, and/or treatingc-Met-associated diseases, such as a cancer. For example, the effect ofpreventing, improving, alleviating, and/or treating a cancer may referto a decrease in cancer cells or cancer tissues, a death of cancer cellsor cancer tissues, an inhibition of cancer cell migration and/orinvasion associated with cancer metastasis, and the like.

PDGF (platelet-derived growth factor) is one of the numerous growthfactors, or proteins that regulate cell growth and division. Inparticular, it plays a significant role in blood vessel formation(angiogenesis), the growth of blood vessels from already-existing bloodvessel tissue. PDGF may be from any mammal, for example, from a primatesuch as human, a monkey, and the like, a rodent such as a rat, a mouse,and the like, but not be limited thereto. For example, PDGF may be atleast one selected from the group consisting of human PDGF (e.g., NCBIAccession No. NP_(—)002598.4, NP_(—)002599.1, NP_(—)057289.1,NP_(—)079484.1, etc.), mouse PDGF (e.g., NCBI Accession No.NP_(—)032834.1, NP_(—)035187.2, NP_(—)064355.1, NP_(—)082200.1, etc.),rat PDGF (e.g., NCBI Accession No. NP_(—)036933.1, NP_(—)113712.1,NP_(—)112607.1, NP_(—)076452.1, etc.), and the like, but not be limitedthereto. PDGF coding gene or mRNA may be at least one selected from thegroup consisting of human PDGF gene (e.g., NCBI Accession No.NM_(—)002607.5, NM_(—)002608.2, NM_(—)016205.2, NM_(—)025208.4, etc.),mouse PDGF gene (e.g., NCBI Accession No. NM_(—)008808.3,NM_(—)011057.3, NM_(—)019971.2, NM_(—)027924.2, etc.), rat PDGF gene(e.g., NCBI Accession No. NM_(—)012801.1, NM_(—)031524.1,NM_(—)031317.1, NM_(—)023962.2, etc.), and the like, but not be limitedthereto.

An embodiment provides a biomarker for predicting an efficacy or aresistance of a c-Met targeting agent, comprising a PDGF protein or agene encoding PDGF.

Another embodiment provides a composition and a kit for predicting anefficacy or a resistance of a c-Met targeting agent, comprising an agentfor measuring the expression level of PDGF protein or the mRNAexpression level of gene encoding PDGF, or a combination thereof.

Another embodiment provides a method for evaluating efficacy of a c-Mettargeting agent on a patient with cancer or the patient's resistance tothe c-Met targeting agent, comprising the steps of:

measuring the expression level of PDGF protein or the mRNA expressionlevel of gene encoding PDGF in a sample obtained from a patient withcancer before administration of a c-Met targeting agent to the patient,and in a sample obtained from the patient after administration of ac-Met targeting agent to the patient,

comparing the expression level of PDGF protein or the mRNA expressionlevel of gene encoding PDGF of the samples obtained before and afteradministration of the c-Met targeting agent, and

evaluating whether the c-Met targeting agent is effective in treatingthe cancer based on a change in the expression level of PDGF protein orthe mRNA expression level of gene encoding PDGF, wherein a significantincrease in the expression level of PDGF protein or the mRNA expressionlevel of gene encoding PDGF in the sample from the patient afteradministration of the c-Met targeting agent, compared to the expressionlevel in the sample before administration of the c-Met targeting agent,indicates that the c-Met targeting agent is not efficacious in treatingthe cancer or the patient has resistance to the c-Met targeting agent.

In an exemplary embodiment, the method for evaluating efficacy of ac-Met targeting agent on a patient with cancer or the patient'sresistance to the c-Met targeting agent, may further comprise the stepof administering the c-Met targeting agent to the subject determined tobe efficacious for treatment.

Another embodiment provides a method for inhibiting c-Met or treatingcancer in a subject, comprising administering a c-Met targeting agent toa subject who has a significant decrease in the PDGF protein level orthe mRNA expression level of gene encoding PDGF in the sample from thesubject after administration of the c-Met targeting agent, compared tothe PDGF protein level or the mRNA expression level in the sample beforeadministration of the c-Met targeting agent.

The method for inhibiting c-Met or treating cancer in a subject mayfurther comprise a step of identifying a subject for application of ac-Met targeting agent, prior to the step of administration. The step ofidentifying may be carried out by performing the steps described in themethod for evaluating efficacy of a c-Met targeting agent on a patientwith cancer or the patient's resistance to the c-Met targeting agent.

As described above, when the expression level of PDGF protein or themRNA expression level of gene encoding PDGF is higher in the biologicalsample, it can be predicted that a resistance to the c-Met targetingagent is present on the biological sample or a patient from who thebiological sample is isolated, or the c-Met targeting agent is notefficacious in treating the cancer. Based thereon, it can be determinedthat the biological sample or a patient from who the biological sampleis isolated is not suitable for applying the c-Met targeting agent

The term “an agent for measuring the expression level of PDGF protein orthe mRNA expression level of gene encoding PDGF” refers to a moleculethat may be used to detect the marker by examining the expression levelof the PDGF marker which is increased when the patient has resistance tothe c-Met targeting agent, for example, marker-specificoligonucleotides, primers, probes, antibodies, aptamers or the like.

As used herein, the term “measurement of mRNA expression level” means aprocess of examining the presence and expression level of mRNA of thePDGF gene in a biological sample through quantification of mRNA.Analysis method thereof includes polymerase chain reaction (PCR; e.g.,reverse transcriptase polymerase chain reaction (RT-PCR), competitiveRT-PCR, Real-time RT-PCR, etc.), hybridization methods (Northernblotting, Microarray, etc.), Taq-based techniques (SAGE, RNA-seq, etc.),DNA chips or the like, but is not limited thereto.

As used herein, the term “measurement of protein expression level” meansa process of examining the presence and expression level of PDGF proteinin a biological sample, and the protein is quantified by using anantibody that specifically binds to the protein of the gene. Analysismethods thereof include immunochromatography, immunohistochemistry,enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA),enzyme immunoassay (EIA), fluorescence immunoassay (FIA), luminescenceimmunoassay (LIA), Western blot, FACS, protein chips and the like, butare not limited thereto.

The agent measuring the mRNA level of the gene may be anoligonucleotide, a pair of primers, or a probe. Since the nucleotidesequence of the gene encoding PDGF is known, those who are skilled inthe art may design primers or probes useful for specifically amplifyingtarget regions of the gene on the basis of the nucleotide sequence.

In an exemplary embodiment, the oligonucleotide may include anoligonucleotide having a nucleic acid sequence complementary to 10 to100, specifically 10 to 50, specifically 10 to 30 consecutive nucleicacid sequence of the gene encoding PDGF. The oligonucleotide having thecomplementary nucleic acid sequence refers to an oligonucleotide havinga sequence hybridizable with the marker gene, and may be anoligonucleotide including a sequence having 80% or more, specifically90% or more, more specifically 95%, for example, 99% or more, or 100%identity to the nucleic acid sequence of the marker gene.

As used herein, the term “primer” means a short nucleic acid sequencehaving a free 3′ hydroxyl group, which is able to form base-pairinginteraction with a complementary template and serves as a starting pointfor replication of the template strand. A primer is able to initiate DNAsynthesis in the presence of a reagent for polymerization (i.e., DNApolymerase or reverse transcriptase) and four different nucleosidetriphosphates at suitable buffers and temperature. PCR amplification maybe performed using sense and antisense primers of polynucleotideencoding PDGF, and the presence of the targeted product may be examinedto evaluate efficacy of, or resistance to, a c-Met targeting agent. PCRconditions and the length of sense and antisense primers may be modifiedon the basis of the methods known in the art.

As used herein, the term “probe” refers to a nucleic acid fragment ofRNA or DNA capable of specifically binding to mRNA, ranging in lengthfrom several to hundreds of bases. The probe may be labeled so as todetect the presence or absence of a specific mRNA. The probe may beprepared in the form of oligonucleotide probe, single stranded DNAprobe, double stranded DNA probe, RNA probe or the like. Hybridizationmay be performed using a probe complementary to the polynucleotideencoding PDGF, and efficacy or resistance of a c-Met targeting agent maybe evaluated by the hybridization result. Selection of suitable probeand hybridization conditions may be modified on the basis of the methodsknown in the art.

The primer or probe may be chemically synthesized using aphosphoramidite solid support method or other widely known methods.These nucleic acid sequences may also be modified using many means knownin the art. Non-limiting examples of such modifications includemethylation, capsulation, replacement of one or more native nucleotideswith analogues thereof, and inter-nucleotide modifications, for example,modifications to uncharged conjugates (e.g., methyl phosphonate,phosphotriester, phosphoroamidate, carbamate, etc.) or chargedconjugates (e.g., phosphorothioate, phosphorodithioate, etc.).

The agent measuring the protein level may be an antibody or an aptamer.

As used herein, the term “antibody” refers to a specific proteinmolecule that indicates an antigenic region. The antibody may refer toan antibody that specifically binds to the marker protein, and includesa monoclonal antibody, a polyclonal antibody, a chimeric antibody, ahuman antibody, a humanized antibody or an antigen-binding fragmentthereof. These antibodies may prepared by a hybridoma method, a phageantibody library method, a genetic recombination method or the like,which is typically used in the art. The term “antigen-binding fragment”refers to a functional fragment of an antibody molecule that retains atleast an antigen-binding function, and it may be exemplified by scFv,(scFv)2, Fab, Fab′ or F(ab′)2, but is not limited thereto.

On the other hand, PDGF or mRNA of a gene encoding PDGF may be used as abiomarker to evaluate efficacy or resistance of a c-Met targeting agentin treating the cancer

In an exemplary embodiment, cancer diseases include all cancers on whicha c-Met targeting agent exhibits its efficacy. The cancer may be a solidcancer or a blood cancer. For example, the cancer may be at least oneselected from the group consisting of squamous cell carcinoma, lungcancer such as small-cell lung cancer, non-small-cell lung cancer,adenocarcinoma of the lung, and squamous cell carcinoma of the lung,peritoneal carcinoma, skin cancer, melanoma in the skin or eyeball,rectal cancer, cancer near the anus, esophagus cancer, small intestinaltumor, endocrine gland cancer, parathyroid cancer, adrenal cancer,soft-tissue sarcoma, urethral cancer, chronic or acute leukemia,lymphocytic lymphoma, hepatoma, gastrointestinal cancer, gastric cancer,pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, livercancer, bladder cancer, hepatocellular adenoma, breast cancer, coloncancer, large intestine cancer, endometrial carcinoma or uterinecarcinoma, salivary gland tumor, kidney cancer, prostate cancer, vulvarcancer, thyroid cancer, head and neck cancers, osteosarcoma, and braincancer, but is not limited thereto. The cancer may be a primary canceror a metastatic cancer. In a particular embodiment, the cancer may be acancer having a resistance to a c-Met targeting agent, for example, ananti-c-Met antibody. The cancer may be a solid cancer such as gastriccancer, lung cancer, kidney cancer, and the like, which has a resistanceto a c-Met targeting agent.

“c-Met” or “c-Met protein” refers to a receptor tyrosine kinase (RTK)which binds hepatocyte growth factor (HGF). c-Met may be derived(obtained) from any species, particularly a mammal, for instance,primates such as human c-Met (e.g., GenBank Accession No. NP_(—)000236),monkey c-Met (e.g., Macaca mulatta, GenBank Accession No.NP_(—)001162100), or rodents such as mouse c-Met (e.g., GenBankAccession No. NP_(—)032617.2), rat c-Met (e.g., GenBank Accession No.NP_(—)113705.1), and the like. The c-Met protein may include apolypeptide encoded by the nucleotide sequence identified as GenBankAccession No. NM_(—)000245, a polypeptide having the amino acid sequenceidentified as GenBank Accession No. NP_(—)000236 or extracellulardomains thereof. The receptor tyrosine kinase c-Met participates invarious mechanisms, such as cancer incidence, metastasis, migration ofcancer cells, invasion of cancer cells, angiogenesis, and the like.

As used herein, the term “c-Met targeting agent” may refer to any agentcapable of recognizing and/or binding to c-Met, degrading c-Met,inhibiting the expression of c-Met, or inhibiting the function of c-Met.For example, the c-Met targeting agent may be an anti-c-Met antibodythat recognizes and/or binds to c-Met or an antigen-binding fragmentthereof. The anti-c-Met antibody or an antigen-binding fragment thereofmay bind to c-Met to induce the degradation thereof c-Met, a receptorfor hepatocyte growth factor (HGF), may be divided into three portions:extracellular, transmembrane, and intracellular. The extracellularportion is composed of an α-subunit and a β-subunit which are linked toeach other through a disulfide bond, and includes a SEMA domainresponsible for binding HGF, a PSI domain (plexin-semaphorins-integrinidentity/homology domain) and an IPT domain (immunoglobulin-like foldshared by plexins and transcriptional factors domain). The SEMA domainof c-Met protein may have the amino acid sequence of SEQ ID NO: 79, andis an extracellular domain that functions to bind HGF. A specific regionof the SEMA domain, that is, a region having the amino acid sequence ofSEQ ID NO: 71, which corresponds to a range from amino acid residues 106to 124 of the amino acid sequence of the SEMA domain (SEQ ID NO: 79), isa loop region between the second and the third propellers within theepitopes of the SEMA domain. This region acts as an epitope for theanti-c-Met antibody.

The term “epitope,” as used herein, refers to an antigenic determinant,a part of an antigen recognized by an antibody. In one embodiment, theepitope may be a region including 5 or more contiguous (consecutive onprimary, secondary (two-dimensional), or tertiary (three-dimensional)structure) amino acid residues within the SEMA domain (SEQ ID NO: 79) ofc-Met protein, for instance, 5 to 19 contiguous amino acid residueswithin the amino acid sequence of SEQ ID NO: 71. For example, theepitope may be a polypeptide having 5 to 19 contiguous amino acidsselected from among partial combinations of the amino acid sequence ofSEQ ID NO: 71, wherein the polypeptide includes at least the aminosequence of SEQ ID NO: 73 (EEPSQ) which serves as an essential elementfor the epitope. For example, the epitope may be a polypeptideincluding, consisting essentially of, or consisting of the amino acidsequence of SEQ ID NO: 71, SEQ ID NO: 72, or SEQ ID NO: 73.

The epitope having the amino acid sequence of SEQ ID NO: 72 correspondsto the outermost part of the loop between the second and thirdpropellers within the SEMA domain of a c-Met protein. The epitope havingthe amino acid sequence of SEQ ID NO: 73 is a site to which the antibodyor antigen-binding fragment according to one embodiment mostspecifically binds.

Thus, the dual-targeting agent to c-Met and EGFR may specifically bindto an epitope which has 5 to 19 contiguous amino acids selected from theamino acid sequence of SEQ ID NO: 71, including SEQ ID NO: 73 (EEPSQ) asan essential element. For example, the dual-targeting agent to c-Met andEGFR may specifically bind to an epitope including the amino acidsequence of SEQ ID NO: 71, SEQ ID NO: 72, or SEQ ID NO: 73.

In one embodiment, the dual-targeting agent to c-Met and EGFR or anantigen-binding fragment thereof may comprise or consist essentially of:

at least one heavy chain complementarity determining region (CDR)selected from the group consisting of (a) a CDR-H1 comprising the aminoacid sequence of SEQ ID NO: 4; (b) a CDR-H2 comprising the amino acidsequence of SEQ ID NO: 5, SEQ ID NO: 2, or an amino acid sequencecomprising 8-19 consecutive amino acids within SEQ ID NO: 2 includingamino acid residues from the 3^(rd) to 10^(th) positions of SEQ ID NO:2; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6,SEQ ID NO: 85, or an amino acid sequence comprising 6-13 consecutiveamino acids within SEQ ID NO: 85 including amino acid residues from the1^(st) to 6^(th) positions of SEQ ID NO: 85, or a heavy chain variableregion comprising the at least one heavy chain complementaritydetermining region;

at least one light chain complementarity determining region (CDR)selected from the group consisting of (a) a CDR-L1 comprising the aminoacid sequence of SEQ ID NO: 7, (b) a CDR-L2 comprising the amino acidsequence of SEQ ID NO: 8, and (c) a CDR-L3 comprising the amino acidsequence of SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 86, or an amino acidsequence comprising 9-17 consecutive amino acids within SEQ ID NO: 89including amino acid residues from the 1^(st) to 9^(th) positions of SEQID NO: 89, or a light chain variable region comprising the at least onelight chain complementarity determining region;

a combination of the at least one heavy chain complementaritydetermining region and at least one light chain complementaritydetermining region; .or

a combination of the heavy chain variable region and the light chainvariable region.

Herein, the amino acid sequences of SEQ ID NOS: 4 to 9 are respectivelyrepresented by following Formulas I to VI, below:

Formula I (SEQ ID NO: 4) Xaa₁-Xaa₂-Tyr-Tyr-Met-Ser,

wherein Xaa₁ is absent or Pro or Ser, and Xaa₂ is Glu or Asp,

Formula II (SEQ ID NO: 5) Arg-Asn-Xaa₃-Xaa₄-Asn-Gly-Xaa₅-Thr,

wherein Xaa₃ is Asn or Lys, Xaa₄ is Ala or Val, and Xaa₅ is Asn or Thr,

Formula III (SEQ ID NO: 6) Asp-Asn-Trp-Leu-Xaa₆-Tyr,

wherein Xaa₆ is Ser or Thr,

Formula IV (SEQ ID NO: 7) Lys-Ser-Ser-Xaa₇-Ser-Leu-Leu-Ala-Xaa₈-Gly-Asn-Xaa₉-Xaa₁₀-Asn-Tyr-Leu-Ala

wherein Xaa₇ is His, Arg, Gln, or Lys, Xaa₈ is Ser or Trp, Xaa₉ is Hisor Gln, and Xaa₁₀ is Lys or Asn,

Formula V (SEQ ID NO: 8) Trp-Xaa₁₁-Ser-Xaa₁₂-Arg-Val-Xaa₁₃

wherein Xaa₁₁ is Ala or Gly, Xaa₁₂ is Thr or Lys, and Xaa₁₃ is Ser orPro, and

Formula VI (SEQ ID NO: 9) Xaa₁₄-Gln-Ser-Tyr-Ser-Xaa₁₅-Pro-Xaa₁₆-Thr

wherein Xaa₁₄ is Gly, Ala, or Gln, Xaa₁₅ is Arg, His, Ser, Ala, Gly, orLys, and Xaa₁₆ is Leu, Tyr, Phe, or Met.

In one embodiment, the CDR-H1 may comprise or consist essentially of anamino acid sequence selected from the group consisting of SEQ ID NOS: 1,22, 23, and 24. The CDR-H2 may comprise or consist essentially of anamino acid sequence selected from the group consisting of SEQ ID NOS: 2,25, and 26. The CDR-H3 may comprise or consist essentially of an aminoacid sequence selected from the group consisting of SEQ ID NOS: 3, 27,28, and 85.

The CDR-L1 may comprise or consist essentially of an amino acid sequenceselected from the group consisting of SEQ ID NOS: 10, 29, 30, 31, 32,33, and 106. The CDR-L2 may comprise or consist essentially of an aminoacid sequence selected from the group consisting of SEQ ID NOS: 11, 34,35, and 36. The CDR-L3 may comprise or consist essentially of an aminoacid sequence selected from the group consisting of SEQ ID NOS: 12, 13,14, 15, 16, 37, 86, and 89.

In another embodiment, the antibody or antigen-binding fragment mayinclude a heavy chain variable region comprising a polypeptide (CDR-H1)including an amino acid sequence selected from the group consisting ofSEQ ID NOS: 1, 22, 23, and 24, a polypeptide (CDR-H2) including an aminoacid sequence selected from the group consisting of SEQ ID NOS: 2, 25,and 26, and a polypeptide (CDR-H3) including an amino acid sequenceselected from the group consisting of SEQ ID NOS: 3, 27, 28, and 85; anda light chain variable region comprising a polypeptide (CDR-L1)including an amino acid sequence selected from the group consisting ofSEQ ID NOS: 10, 29, 30, 31, 32, 33 and 106, a polypeptide (CDR-L2)including an amino acid sequence selected from the group consisting ofSEQ ID NOS: 11, 34, 35, and 36, and a polypeptide (CDR-L3) including anamino acid sequence selected from the group consisting of SEQ ID NOS 12,13, 14, 15, 16, 37, 86, and 89.

In one embodiment of the dual-targeting agent to c-Met and EGFR orantigen-binding fragment, the variable region of the heavy chainincludes the amino acid sequence of SEQ ID NO: 17, 74, 87, 90, 91, 92,93, or 94 and the variable region of the light chain includes the aminoacid sequence of SEQ ID NO: 18, 19, 20, 21, 75, 88, 95, 96, 97, 98, 99,or 107.

Animal-derived antibodies produced by immunizing non-immune animals witha desired antigen generally invoke immunogenicity when injected tohumans for the purpose of medical treatment, and thus chimericantibodies have been developed to inhibit such immunogenicity. Chimericantibodies are prepared by replacing constant regions of animal-derivedantibodies that cause an anti-isotype response with constant regions ofhuman antibodies by genetic engineering. Chimeric antibodies areconsiderably improved in terms of anti-isotype response compared toanimal-derived antibodies, but animal-derived amino acids still havevariable regions, so that chimeric antibodies have side effects withrespect to a potential anti-idiotype response. Humanized antibodies havebeen developed to reduce such side effects. Humanized antibodies areproduced by grafting complementarity determining regions (CDR) whichserve an important role in antigen binding in variable regions ofchimeric antibodies into a human antibody framework.

In using CDR grafting to produce humanized antibodies, choosing whichoptimized human antibodies to use for accepting CDRs of animal-derivedantibodies is critical. Antibody databases, analysis of a crystalstructure, and technology for molecule modeling are used. However, evenwhen the CDRs of animal-derived antibodies are grafted to the mostoptimized human antibody framework, amino acids positioned in aframework of the animal-derived CDRs affecting antigen binding arepresent. Therefore, in many cases, antigen binding affinity is notmaintained, and thus application of additional antibody engineeringtechnology for recovering the antigen binding affinity is necessary.

The anti c-Met antibodies may be, but are not limited to, animalantibodies (e.g., mouse-derived antibodies), chimeric antibodies (e.g.,mouse-human chimeric antibodies), humanized antibodies, or humanantibodies. The antibodies or antigen-binding fragments thereof may beisolated from a living body or non-naturally occurring. The antibodiesor antigen-binding fragments thereof may be synthetic or recombinant.The antibody may be monoclonal.

An intact antibody includes two full-length light chains and twofull-length heavy chains, in which each light chain is linked to a heavychain by disulfide bonds. The antibody has a heavy chain constant regionand a light chain constant region. The heavy chain constant region is ofa gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε) type, whichmay be further categorized as gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3),gamma 4 (γ4), alpha 1 (α1), or alpha 2 (α2). The light chain constantregion is of either a kappa (κ) or lambda (λ) type.

As used herein, the term “heavy chain” refers to full-length heavychain, and fragments thereof, including a variable region V_(H) thatincludes amino acid sequences sufficient to provide specificity toantigens, and three constant regions, C_(H1), C_(H2), and C_(H3), and ahinge. The term “light chain” refers to a full-length light chain andfragments thereof, including a variable region V_(L) that includes aminoacid sequences sufficient to provide specificity to antigens, and aconstant region C_(L).

The term “complementarity determining region” (“CDR”) refers to an aminoacid sequence found in a hyper variable region of a heavy chain or alight chain of immunoglobulin. The heavy and light chains mayrespectively include three CDRs (CDRH1, CDRH2, and CDRH3; and CDRL1,CDRL2, and CDRL3). The CDR may provide contact residues that play animportant role in the binding of antibodies to antigens or epitopes. Theterms “specifically binding” and “specifically recognized” are wellknown to one of ordinary skill in the art, and indicate that an antibodyand an antigen specifically interact with each other to lead to animmunological activity.

The term “antigen-binding fragment” used herein refers to fragments ofan intact immunoglobulin including portions of a polypeptide includingantigen-binding regions having the ability to specifically bind to theantigen. In a particular embodiment, the antigen-binding fragment may bescFv, (scFv)₂, scFvFc, Fab, Fab′, or F(ab′)₂, but is not limitedthereto.

Among the antigen-binding fragments, Fab that includes light chain andheavy chain variable regions, a light chain constant region, and a firstheavy chain constant region C_(H1), has one antigen-binding site.

The Fab′ fragment is different from the Fab fragment, in that Fab′includes a hinge region with at least one cysteine residue at theC-terminal of C_(H1).

The F(ab′)₂ antibody is formed through disulfide bridging of thecysteine residues in the hinge region of the Fab′ fragment.

Fv is the smallest antibody fragment with only a heavy chain variableregion and a light chain variable region. Recombination techniques ofgenerating the Fv fragment are widely known in the art.

Two-chain Fv includes a heavy chain variable region and a light chainregion which are linked by a non-covalent bond. Single-chain Fvgenerally includes a heavy chain variable region and a light chainvariable region which are linked by a covalent bond via a peptide linkeror linked at the C-terminals to have a dimer structure like thetwo-chain Fv. The peptide linker may be the same as described above,including, but not limited to, those having an amino acid length of 1 to100, or 2 to 50, and any kinds of amino acids may be included withoutany restrictions.

The antigen-binding fragments may be obtained using protease (forexample, the Fab fragment may be obtained by restricted cleavage of awhole antibody with papain, and the F(ab′)₂ fragment may be obtained bycleavage with pepsin), or may be prepared by using a geneticrecombination technique.

The term “hinge region,” as used herein, refers to a region between CH1and CH2 domains within the heavy chain of an antibody which functions toprovide flexibility for the antigen-binding site.

When an animal antibody undergoes a chimerization process, the IgG1hinge of animal origin is replaced with a human IgG1 hinge or IgG2 hingewhile the disulfide bridges between two heavy chains are reduced fromthree to two in number. In addition, an animal-derived IgG1 hinge isshorter than a human IgG1 hinge. Accordingly, the rigidity of the hingeis changed. Thus, a modification of the hinge region may bring about animprovement in the antigen binding efficiency of the humanized antibody.The modification of the hinge region through amino acid deletion,addition, or substitution is well-known to those skilled in the art.

In one embodiment, the dual-targeting agent to c-Met and EGFR or anantigen-binding fragment thereof may be modified by any combination ofdeletion, insertion, addition, or substitution of at least one aminoacid residue on the amino acid sequence of the hinge region so that itexhibit enhanced antigen-binding efficiency. For example, the antibodymay include a hinge region including the amino acid sequence of SEQ IDNO: 100(U7-HC6), 101(U6-HC7), 102(U3-HC9), 103(U6-HC8), or 104(U8-HC5),or a hinge region including the amino acid sequence of SEQ ID NO: 105(non-modified human hinge). In particular, the hinge region has theamino acid sequence of SEQ ID NO: 100 or 101.

In one embodiment, the dual-targeting agent to c-Met and EGFR may be amonoclonal antibody. The monoclonal antibody may be produced by thehybridoma cell line deposited with Accession No. KCLRF-BP-00220, whichbinds specifically to the extracellular region of c-Met protein (referto Korean Patent Publication No. 2011-0047698, the entire disclosure ofwhich is hereby incorporated by reference). The dual-targeting agent toc-Met and EGFR may include all the antibodies defined in Korean PatentPublication No. 2011-0047698.

In the c-Met antibody or an antigen-binding fragment thereof, the restportion of the light chain and the heavy chain portion except the CDRs,the light chain variable region, and the heavy chain variable region asdefined above, for example, the light chain constant region and theheavy chain constant region, may be from any subtype of immunoglobulin(e.g., IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), IgM, and the like).

By way of further example, the dual-targeting agent to c-Met and EGFR orthe antibody fragment may comprise or consist essentially of:

a heavy chain including the amino acid sequence selected from the groupconsisting of the amino acid sequence of SEQ ID NO: 62 (wherein theamino acid sequence from amino acid residues from the 1^(st) to 17^(th)positions is a signal peptide), or the amino acid sequence from the18^(th) to 462^(nd) positions of SEQ ID NO: 62, the amino acid sequenceof SEQ ID NO: 64 (wherein the amino acid sequence from the 1^(st) to17^(th) positions is a signal peptide), the amino acid sequence from the18^(th) to 461^(st) positions of SEQ ID NO: 64, the amino acid sequenceof SEQ ID NO: 66 (wherein the amino acid sequence from the 1^(st) to17^(th) positions is a signal peptide), and the amino acid sequence fromthe 18^(th) to 460^(th) positions of SEQ ID NO: 66; and

a light chain including the amino acid sequence selected from the groupconsisting of the amino acid sequence of SEQ ID NO: 68 (wherein theamino acid sequence from the 1^(st) to 20^(th) positions is a signalpeptide), the amino acid sequence from the 21^(st) to 240^(th) positionsof SEQ ID NO: 68, the amino acid sequence of SEQ ID NO: 70 (wherein theamino acid sequence from the 1^(st) to 20^(th) positions is a signalpeptide), the amino acid sequence from the 21^(st) to 240^(th) positionsof SEQ ID NO: 70, and the amino acid sequence of SEQ ID NO: 108.

For example, the dual-targeting agent to c-Met and EGFR may be selectedfrom the group consisting of:

an antibody including a heavy chain including the amino acid sequence ofSEQ ID NO: 62 or the amino acid sequence from the 18^(th) to 462^(nd)positions of SEQ ID NO: 62 and a light chain including the amino acidsequence of SEQ ID NO: 68 or the amino acid sequence from the 21^(st) to240^(th) positions of SEQ ID NO: 68;

an antibody including a heavy chain including the amino acid sequence ofSEQ ID NO: 64 or the amino acid sequence from the 18^(th) to 461^(st)positions of SEQ ID NO: 64 and a light chain including the amino acidsequence of SEQ ID NO: 68 or the amino acid sequence from the 21^(st) to240^(th) positions of SEQ ID NO: 68;

an antibody including a heavy chain including the amino acid sequence ofSEQ ID NO: 66 or the amino acid sequence from the 18^(th) to 460^(th)positions of SEQ ID NO: 66 and a light chain including the amino acidsequence of SEQ ID NO: 68 or the amino acid sequence from the 21^(st) to240^(th) positions of SEQ ID NO: 68;

an antibody including a heavy chain including the amino acid sequence ofSEQ ID NO: 62 or the amino acid sequence from the 18^(th) to 462^(nd)positions of SEQ ID NO: 62 and a light chain including the amino acidsequence of SEQ ID NO: 70 or the amino acid sequence from the 21^(st) to240^(th) positions of SEQ ID NO: 70;

an antibody including a heavy chain including the amino acid sequence ofSEQ ID NO: 64 or the amino acid sequence from the 18^(th) to 461^(st)positions of SEQ ID NO: 64 and a light chain including the amino acidsequence of SEQ ID NO: 70 or the amino acid sequence from the 21^(st) to240^(th) positions of SEQ ID NO: 70;

an antibody including a heavy chain including the amino acid sequence ofSEQ ID NO: 66 or the amino acid sequence from the 18^(th) to 460^(th)positions of SEQ ID NO: 66 and a light chain including the amino acidsequence of SEQ ID NO: 70 or the amino acid sequence from the 21^(st) to240^(th) positions of SEQ ID NO: 70;

an antibody including a heavy chain including the amino acid sequence ofSEQ ID NO: 62 or the amino acid sequence from the 18^(th) to 462^(nd)positions of SEQ ID NO: 62 and a light chain including the amino acidsequence of SEQ ID NO: 108;

an antibody including a heavy chain including the amino acid sequence ofSEQ ID NO: 64 or the amino acid sequence from the 18^(th) to 461^(st)positions of SEQ ID NO: 64 and a light chain including the amino acidsequence of SEQ ID NO: 108; and

an antibody including a heavy chain including the amino acid sequence ofSEQ ID NO: 66 or the amino acid sequence from the 18^(th) to 460^(th)positions of SEQ ID NO: 66 and a light chain including the amino acidsequence of SEQ ID NO: 108.

The polypeptide of SEQ ID NO: 70 is a light chain including human kappa(κ) constant region, and the polypeptide with the amino acid sequence ofSEQ ID NO: 68 is a polypeptide obtained by replacing histidine atposition 62 (corresponding to position 36 of SEQ ID NO: 68 according tokabat numbering) of the polypeptide with the amino acid sequence of SEQID NO: 70 with tyrosine. The production yield of the antibodies may beincreased by the replacement. The polypeptide with the amino acidsequence of SEQ ID NO: 108 is a polypeptide obtained by replacing serineat position 32 (position 27e according to kabat numbering in the aminoacid sequence from amino acid residues 21 to 240 of SEQ ID NO: 68;positioned within CDR-L1) with tryptophan. By such replacement,antibodies and antibody fragments including such sequences exhibitsincreased activities, such as c-Met biding affinity, c-Met degradationactivity, and Akt phosphorylation inhibition.

In another embodiment, the dual-targeting agent to c-Met and EGFR mayinclude a light chain complementarity determining region including theamino acid sequence of SEQ ID NO: 106, a light chain variable regionincluding the amino acid sequence of SEQ ID NO: 107, or a light chainincluding the amino acid sequence of SEQ ID NO: 108.

As used herein, “patient” refers to an animal including a monkey, cow,horse, sheep, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbitor guinea pig as well as human, but is not limited thereto. In oneembodiment, it may refer to a mammal, and in another embodiment, it mayrefer to a human.

As used herein, “kit” may include other elements essential for measuringthe expression level of PDGF protein or the mRNA expression level ofgene encoding PDGF, in addition to the agent for measuring theexpression level of PDGF protein or the mRNA expression level of geneencoding PDGF.

In one embodiment, the kit may be a kit including essential elementsrequired for performing PCR, and to this end, the kit may include testtubes or other suitable containers, reaction buffers (varying in pH andmagnesium concentrations), deoxynucleotides (dNTPs), enzymes such asTaq-polymerase and reverse transcriptase, DNase, RNase inhibitor, DEPCwater, and sterile water, in addition to a pair of primers specific tothe marker gene encoding PDGF that are designed by those skilled in theart.

In another embodiment, the kit may be a kit including essential elementsrequired for performing a DNA chip, and to this end, the kit may includea base plate, onto which cDNAs corresponding to genes or fragmentsthereof are attached, and the base plate may include cDNA correspondingto a quantification control gene or a fragment thereof.

In still another embodiment, the kit may be a kit for measuring the PDGFprotein expression level, and may include a matrix, a suitable buffersolution, a coloring enzyme, or a secondary antibody labeled with afluorescent substance, a coloring substrate or the like for theimmunological detection of antibody. As for the matrix, a nitrocellulosemembrane, a 96-well plate made of polyvinyl resin, a 96-well plate madeof polystyrene resin, and a slide glass may be used. As for the coloringenzyme, peroxidase and alkaline phosphatase may be used. As for thefluorescent substance, FITC, RITC or the like may be used, and as forthe coloring substrate solution, ABTS(2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid)), OPD(o-phenylenediamine), or TMB (tetramethyl benzidine) or the like may beused. However, they are not limited thereto.

Analysis methods for measuring mRNA levels include RT-PCR, competitiveRT-PCR, real-time RT-PCR, RNase protection assay, Northern blotting andDNA chip assay, but are not limited thereto. With the detection methods,the mRNA expression levels may be compared in the samples of the patientbefore and after administration of an c-Met targeting agent, andefficacy or resistance to a c-Met targeting agent may be evaluated bydetermining whether mRNA expression levels of the gene encoding PDGFhave significantly decreased or increased.

The mRNA expression levels may be measured by a variety of polymerasechain reaction techniques, hybridization methods or DNA chip assaysusing primers being specific to the gene encoding PDGF.

In one embodiment, the resulting products of RT-PCR may beelectrophoresed, and patterns and thicknesses of bands may be analyzedto determine the expression and levels of mRNA from the gene encodingPDGF while comparing the mRNA expression and levels with those of acontrol group, thereby evaluating efficacy or resistance to a c-Mettargeting agent.

In another embodiment, the DNA chip may be used, in which the geneencoding PDGF or a nucleic acid corresponding to a fragment thereof isanchored at high density to a glass-like base plate. mRNA may beisolated from the sample, and a cDNA probe labeled with a fluorescentsubstance at its end or internal region may be prepared, followed byhybridization with the DNA chip, thereby evaluating efficacy orresistance to a c-Met targeting agent.

Analysis methods for measuring protein levels includeimmunochromatography, immunohistochemistry, ELISA, radioimmunoassay,enzyme immunoassay, fluorescence immunoassay, luminescence immunoassay,Western blot, FACS, protein chips or the like, but are not limitedthereto. With the analysis methods, the amount of formedantigen-antibody complexes may be compared in the samples of the patientbefore and after administration of c-Met targeting agent, and efficacyof, or resistance to, a c-Met targeting agent may be evaluated bydetermining whether protein expression levels of the marker gene havesignificantly decreased or increased.

As used herein, the term “antigen-antibody complexes” refers to bindingproducts of the PDGF to an antibody specific thereto. The amount offormed antigen-antibody complexes may be quantitatively determined bymeasuring the signal intensity of a detection label.

In one embodiment, the protein expression levels may be measured byELISA. ELISA includes a variety of ELISA methods such as direct ELISAusing a labeled antibody recognizing an antigen immobilized on a solidsupport, indirect ELISA using a labeled antibody recognizing a captureantibody forming complexes with an antigen immobilized on a solidsupport, direct sandwich ELISA using another labeled antibodyrecognizing an antigen in an antigen-antibody complex immobilized on asolid support, and indirect sandwich ELISA, in which another labeledantibody recognizing an antigen in an antigen-antibody compleximmobilized on a solid support is reacted, and then a secondary labeledantibody recognizing the another labeled antibody may be used. Moreparticularly, the protein expression levels may be detected by sandwichELISA, where a sample is reacted with an antibody immobilized on a solidsupport, and the resulting antigen-antibody complexes are detected byadding a labeled antibody recognizing the antigen of antigen-antibodycomplex, followed by enzymatic color development, or by adding asecondary labeled antibody specific to the antibody which recognizes theantigen of the antigen-antibody complex, followed by enzymaticdevelopment. The efficacy of, or resistance to, a c-Met targeting agentmay be evaluated by measuring the degree of complex formation of PDGFprotein and antibody.

In another embodiment, the protein expression levels may be measured byWestern blotting using one or more antibodies against PDGF. In thismethod, total proteins may be isolated from a sample, electrophoresed tobe separated according to size, transferred onto a nitrocellulosemembrane, and reacted with an antibody. The amount of producedantigen-antibody complexes may be measured using a labeled antibody toevaluate efficacy of, or resistance to, a c-Met targeting agent.

In still another embodiment, the protein expression levels may bemeasured by immunohistostaining using one or more antibodies againstPDGF. In this method, cancer tissues are collected and fixed before andafter administration of the c-Met targeting agent, and thenparaffin-embedded blocks are prepared according to a widely knownmethod. The blocks may cut into sections being several μm in thickness,and attach to glass slides to be reacted with one or more selected PDGFantibodies according to the known method. Subsequently, the unreactedantibodies may be washed, and the reacted antibodies labeled with oneselected from the above mentioned detection labels, and then the labeledantibodies may be observed under a microscope.

In still another embodiment, the protein expression levels may bemeasured using a protein chip in which one or more antibodies againstPDGF are arranged and fixed at a high density at predetermined positionson a substrate. In this method of sample analysis using the proteinchip, proteins may be separated from a sample, and the separatedproteins may be hybridized with the protein chip to form anantigen-antibody complex, which is then read to examine the presence orexpression level of the protein, thereby evaluating efficacy orresistance to a c-Met targeting agent.

EXAMPLES

Hereafter, the present invention will be described in detail byexamples.

The following examples are intended merely to illustrate the inventionand are not construed to restrict the invention.

1.1. Production of “AbF46”, a Mouse Antibody to c-Met

1.1.1. Immunization of Mouse

To obtain immunized mice necessary for the development of a hybridomacell line, each of five BALB/c mice (Japan SLC, Inc.), 4 to 6 weeks old,was intraperitoneally injected with a mixture of 100 μg of humanc-Met/Fc fusion protein (R&D Systems) and one volume of completeFreund's adjuvant. Two weeks after the injection, a secondintraperitoneal injection was conducted on the same mice with a mixtureof 50 μg of human c-Met/Fc protein and one volume of incomplete Freund'sadjuvant. One week after the second immunization, the immune responsewas finally boosted. Three days later, blood was taken from the tails ofthe mice and the sera were 1/1000 diluted in PBS and used to examine atiter of antibody to c-Met by ELISA. Mice found to have a sufficientantibody titer were selected for use in the cell fusion process.

1.1.2. Cell Fusion and Production of Hybridoma

Three days before cell fusion, BALB/c mice (Japan SLC, Inc.) wereimmunized with an intraperitoneal injection of a mixture of 50 μg ofhuman c-Met/Fc fusion protein and one volume of PBS. The immunized micewere anesthetized before excising the spleen from the left half of thebody. The spleen was meshed to separate splenocytes which were thensuspended in a culture medium (DMEM, GIBCO, Invitrogen). The cellsuspension was centrifuged to recover the cell layer. The splenocytesthus obtained (1×10⁸ cells) were mixed with myeloma cells (Sp2/0) (1×10⁸cells), followed by spinning to give a cell pellet. The cell pellet wasslowly suspended, treated with 45% polyethylene glycol (PEG) (1 mL) inDMEM for 1 min at 37° C., and supplemented with 1 mL of DMEM. To thecells was added 10 mL of DMEM over 10 min, after which incubation wasconducted in a water bath at 37° C. for 5 min. Then the cell volume wasadjusted to 50 mL before centrifugation. The cell pellet thus formed wasresuspended at a density of 1˜2×10⁵ cells/mL in a selection medium (HATmedium) and 0.1 mL of the cell suspension was allocated to each well of96-well plates which were then incubated at 37° C. in a CO₂ incubator toestablish a hybridoma cell population.

1.1.3. Selection of Hybridoma Cells Producing Monoclonal Antibodies toc-Met Protein

From the hybridoma cell population established in Reference Example1.1.2, hybridoma cells which showed a specific response to c-Met proteinwere screened by ELISA using human c-Met/Fc fusion protein and human Fcprotein as antigens.

Human c-Met/Fc fusion protein was seeded in an amount of 50 μL (2μg/mL)/well to microtiter plates and allowed to adhere to the surface ofeach well. The antibody that remained unbound was removed by washing.For use in selecting the antibodies that do not bind c-Met but recognizeFc, human Fc protein was attached to the plate surface in the samemanner.

The hybridoma cell culture obtained in Reference Example 1.1.2 was addedin an amount of 50 μL to each well of the plates and incubated for 1hour. The cells remaining unreacted were washed out with a sufficientamount of Tris-buffered saline and Tween 20 (TBST). Goat anti-mouseIgG-horseradish peroxidase (HRP) was added to the plates and incubatedfor 1 hour at room temperature. The plates were washed with a sufficientamount of TBST, followed by reacting the peroxidase with a substrate(OPD). Absorbance at 450 nm was measured on an ELISA reader.

Hybridoma cell lines which secrete antibodies that specifically andstrongly bind to human c-Met but not human Fc were selected repeatedly.From the hybridoma cell lines obtained by repeated selection, a singleclone producing a monoclonal antibody was finally separated by limitingdilution. The single clone of the hybridoma cell line producing themonoclonal antibody was deposited with the Korean Cell Line ResearchFoundation, an international depository authority located atYungun-Dong, Jongno-Gu, Seoul, Korea, on Oct. 6, 2009, with AccessionNo. KCLRF-BP-00220 according to the Budapest Treaty (refer to KoreanPatent Laid-Open Publication No. 2011-0047698).

1.1.4. Production and Purification of Monoclonal Antibody

The hybridoma cell line obtained in Reference Example 1.1.3 was culturedin a serum-free medium, and the monoclonal antibody (AbF46) was producedand purified from the cell culture.

First, the hybridoma cells cultured in 50 mL of a medium (DMEM)supplemented with 10% (v/v) FBS were centrifuged and the cell pellet waswashed twice or more with 20 mL of PBS to remove the FBS therefrom.Then, the cells were resuspended in 50 mL of DMEM and incubated for 3days at 37° C. in a CO₂ incubator.

After the cells were removed by centrifugation, the supernatant wasstored at 4° C. before use or immediately used for the separation andpurification of the antibody. An AKTA system (GE Healthcare) equippedwith an affinity column (Protein G agarose column; Pharmacia, USA) wasused to purify the antibody from 50 to 300 mL of the supernatant,followed by concentration with an filter (Amicon). The antibody in PBSwas stored before use in the following examples.

1.2. Construction of chAbF46, a Chimeric Antibody to c-Met

A mouse antibody is apt to elicit immunogenicity in humans. To solvethis problem, chAbF46, a chimeric antibody, was constructed from themouse antibody AbF46 produced in Experimental Example 1.1.4 by replacingthe constant region, but not the variable region responsible forantibody specificity, with an amino sequence of the human IgG1 antibody.

In this regard, a gene was designed to include the nucleotide sequenceof “EcoRI-signal sequence-VH-NheI-CH-TGA-XhoI” (SEQ ID NO: 38) for aheavy chain and the nucleotide sequence of “EcoRI-signalsequence-VL-BsiWI-CL-TGA-XhoI” (SEQ ID NO: 39) for a light chain andsynthesized. Then, a DNA fragment having the heavy chain nucleotidesequence (SEQ ID NO: 38) and a DNA fragment having the light chainnucleotide sequence (SEQ ID NO: 39) were digested with EcoRI (NEB,R0101S) and XhoI (NEB, R0146S) before cloning into a pOptiVEC™-TOPO TACloning Kit enclosed in an OptiCHO™ Antibody Express Kit (Cat no.12762-019, Invitrogen), and a pcDNA™3.3-TOPO TA Cloning Kit (Cat no.8300-01), respectively.

Each of the constructed vectors was amplified using Qiagen Maxiprep kit(Cat no. 12662), and a transient expression was performed usingFreestyle™ MAX 293 Expression System (invitrogen). 293 F cells were usedfor the expression and cultured in FreeStyle™ 293 Expression Medium in asuspension culture manner. One day prior to the transient expression,the cells were provided in the concentration of 5×10⁵ cells/ml, andafter 24 hours, when the cell number reached to 1×10⁶ cells/ml, thetransient expression was performed. A transfection was performed by aliposomal reagent method using Freestyle™ MAX reagent (Invitrogen),wherein in a 15 ml tube, the DNA was provided in the mixture ratio of1:1 (heavy chain DNA:light chain DNA) and mixed with 2 ml of OptiPro™SFM (Invitrogen) (A), and in another 15 ml tube, 100 ul (microliter) ofFreestyle™ MAX reagent and 2 ml of OptiPro™ SFM were mixed (B), followedby mixing (A) and (B) and incubating for 15 minutes. The obtainedmixture was slowly mixed with the cells provided one day before thetransient expression. After completing the transfection, the cells wereincubated in 130 rpm incubator for 5 days under the conditions of 37°C., 80% humidity, and 8% CO₂.

Afterwards, the cells were incubated in DMEM supplemented with 10% (v/v)FBS for 5 hours at 37° C. under a 5% CO₂ condition and then in FBS-freeDMEM for 48 hours at 37° C. under a 5% CO₂ condition.

After centrifugation, the supernatant was applied to AKTA prime (GEHealthcare) to purify the antibody. In this regard, 100 mL of thesupernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equippedwith a Protein A column (GE healthcare, 17-0405-03), followed by elutionwith an IgG elution buffer (Thermo Scientific, 21004). The buffer wasexchanged with PBS to purify a chimeric antibody AbF46 (hereinafterreferred to as “chAbF46”).

1.3. Construction of Humanized Antibody huAbF46 from Chimeric AntibodychAbF46

1.3.1. Heavy Chain Humanization

To design two domains H1-heavy and H3-heavy, human germline genes whichshare the highest identity/homology with the VH gene of the mouseantibody AbF46 purified in Reference Example 1.2 were analyzed. An IgBLAST (www.ncbi.nlm.nih.gov/igblast/) result revealed that VH3-71 has anidentity/identity/homology of 83% at the amino acid level. CDR-H1,CDR-H2, and CDR-H3 of the mouse antibody AbF46 were defined according toKabat numbering. A design was made to introduce the CDR of the mouseantibody AbF46 into the framework of VH3-71. Back mutations to the aminoacid sequence of the mouse AbF46 were conducted at positions 30 (S→T),48 (V→L), 73 (D→N), and 78 (T→L). Then, H1 was further mutated atpositions 83 (R→K) and 84 (A→T) to finally establish H1-heavy (SEQ IDNO: 40) and H3-heavy (SEQ ID NO: 41).

For use in designing H4-heavy, human antibody frameworks were analyzedby a BLAST search. The result revealed that the VH3 subtype, known to bemost stable, is very similar in framework and sequence to the mouseantibody AbF46. CDR-H1, CDR-H2, and CDR-H3 of the mouse antibody AbF46were defined according to Kabat numbering and introduced into the VH3subtype to construct H4-heavy (SEQ ID NO: 42).

1.3.2. Light Chain Humanization

To design two domains H1-light (SEQ ID NO: 43) and H2-light (SEQ ID NO:44), human germline genes which share the highest identity/homology withthe VH gene of the mouse antibody AbF46 were analyzed. An Ig BLASTsearch result revealed that VK4-1 has an identity/homology of 75% at theamino acid level. CDR-L1, CDR-L2, and CDR-L3 of the mouse antibody AbF46were defined according to Kabat numbering. A design was made tointroduce the CDR of the mouse antibody AbF46 into the framework ofVK4-1. Back mutations to the amino acid sequence of the mouse AbF46 wereconducted at positions 36 (Y→H), 46 (L→M), and 49 (Y→I). Only one backmutation was conducted at position 49 (Y→I) on H2-light.

To design H3-light (SEQ ID NO: 45), human germline genes which share thehighest identity/homology with the VL gene of the mouse antibody AbF46were analyzed by a search for BLAST. As a result, VK2-40 was selected.VL and VK2-40 of the mouse antibody AbF46 were found to have aidentity/homology of 61% at an amino acid level. CDR-L1, CDR-L2, andCDR-L3 of the mouse antibody were defined according to Kabat numberingand introduced into the framework of VK4-1. Back mutations wereconducted at positions 36 (Y→H), 46 (L→M), and 49 (Y→I) on H3-light.

For use in designing H4-light (SEQ ID NO: 46), human antibody frameworkswere analyzed. A Blast search revealed that the Vk1 subtype, known to bethe most stable, is very similar in framework and sequence to the mouseantibody AbF46. CDR-L1, CDR-L2, and CDR-L3 of the mouse antibody AbF46were defined according to Kabat numbering and introduced into the Vk1subtype. Back mutations were conducted at positions 36 (Y→H), 46 (L→M),and 49 (Y→I) on H4-light.

Thereafter, DNA fragments having the heavy chain nucleotide sequences(H1-heavy: SEQ ID NO: 47, H3-heavy: SEQ ID NO: 48, H4-heavy: SEQ ID NO:49) and DNA fragments having the light chain nucleotide sequences(H1-light: SEQ ID NO: 50, H2-light: SEQ ID NO: 51, H3-light: SEQ ID NO:52, H4-light: SEQ ID NO: 53) were digested with EcoRI (NEB, R0101S) andXhoI (NEB, R0146S) before cloning into a pOptiVEC™-TOPO TA Cloning Kitenclosed in an OptiCHO™ Antibody Express Kit (Cat no. 12762-019,Invitrogen) and a pcDNA™3.3-TOPO TA Cloning Kit (Cat no. 8300-01),respectively, so as to construct recombinant vectors for expressing ahumanized antibody.

Each of the constructed vectors was amplified using Qiagen Maxiprep kit(Cat no. 12662), and a transient expression was performed usingFreestyle™ MAX 293 Expression System (invitrogen). 293 F cells were usedfor the expression and cultured in FreeStyle™ 293 Expression Medium in asuspension culture manner. At one day before the transient expression,the cells were provided in the concentration of 5×10⁵ cells/ml, andafter 24 hours, when the cell number reached to 1×10⁶ cells/ml, thetransient expression was performed. A transfection was performed by aliposomal reagent method using Freestyle™ MAX reagent (Invitrogen),wherein in a 15 ml tube, the DNA was provided in the mixture ratio of1:1 (heavy chain DNA:light chain DNA) and mixed with 2 ml of OptiPro™SFM (Invitrogen) (A), and in another 15 ml tube, 100 ul (microliter) ofFreestyle™ MAX reagent and 2 ml of OptiPro™ SFM were mixed (B), followedby mixing (A) and (B) and incubating for 15 minutes. The obtainedmixture was slowly mixed with the cells provided one day before thetransient expression. After completing the transfection, the cells wereincubated in 130 rpm incubator for 5 days under the conditions of 37°C., 80% humidity, and 8% CO₂.

After centrifugation, the supernatant was applied to AKTA prime (GEHealthcare) to purify the antibody. In this regard, 100 mL of thesupernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equippedwith a Protein A column (GE healthcare, 17-0405-03), followed by elutionwith an IgG elution buffer (Thermo Scientific, 21004). The buffer wasexchanged with PBS to purify a humanized antibody AbF46 (hereinafterreferred to as “huAbF46”). The humanized antibody huAbF46 used in thefollowing examples included a combination of H4-heavy (SEQ ID NO: 42)and H4-light (SEQ ID NO: 46).

1.4. Construction of scFv Library of huAbF46 Antibody

For use in constructing an scFv of the huAbF46 antibody from the heavyand light chain variable regions of the huAbF46 antibody, a gene wasdesigned to have the structure of “VH-linker-VL” for each of the heavyand the light chain variable region, with the linker including the aminoacid sequence “GLGGLGGGGSGGGGSGGSSGVGS” (SEQ ID NO: 54). Apolynucleotide sequence (SEQ ID NO: 55) encoding the designed scFv ofhuAbF46 was synthesized in Bioneer and an expression vector for thepolynucleotide had the nucleotide sequence of SEQ ID NO: 56.

After expression, the product was found to exhibit specificity to c-Met.

1.5. Construction of Library Genes for Affinity Maturation

1.5.1. Selection of Target CDRs and Synthesis of Primers

The affinity maturation of huAbF46 was achieved. First, sixcomplementary determining regions (CDRs) were defined according to Kabatnumbering. The CDRs are given in Table 2, below.

TABLE 2 CDR Amino Acid Sequence CDR-H1 DYYMS (SEQ ID NO: 1) CDR-H2FIRNKANGYTTEYSASVKG (SEQ ID NO: 2) CDR-H3 DNWFAY (SEQ ID NO: 3) CDR-L1KSSQSLLASGNQNNYLA (SEQ ID NO: 10) CDR-L2 WASTRVS (SEQ ID NO: 11) CDR-L3QQSYSAPLT (SEQ ID NO: 12)

For use in the introduction of random sequences into the CDRs of theantibody, primers were designed as follows. Conventionally, N codonswere utilized to introduce bases at the same ratio (25% A, 25% G, 25% C,25% T) into desired sites of mutation. In this experiment, theintroduction of random bases into the CDRs of huAbF46 was conducted insuch a manner that, of the three nucleotides per codon in the wild-typepolynucleotide encoding each CDR, the first and second nucleotidesconserved over 85% of the entire sequence while the other threenucleotides were introduced at the same percentage (each 5%) and thatthe same possibility was imparted to the third nucleotide (33% G, 33% C,33% T).

1.5.2. Construction of a Library of huAbF46 Antibodies and Affinity forc-Met

The construction of antibody gene libraries through the introduction ofrandom sequences was carried out using the primers synthesized in thesame manner as in Reference Example 1.5.1. Two PCR products wereobtained using a polynucleotide covering the scFV of huAbF46 as atemplate, and were subjected to overlap extension PCR to give scFvlibrary genes for huAbF46 antibodies in which only desired CDRs weremutated. Libraries targeting each of the six CDRs prepared from the scFVlibrary genes were constructed.

The affinity for c-Met of each library was compared to that of thewildtype. Most libraries were lower in affinity for c-Met, compared tothe wild-type. The affinity for c-Met was retained in some mutants.

1.6. Selection of Antibody with Improved Affinity from Libraries

After maturation of the affinity of the constructed libraries for c-Met,the nucleotide sequence of scFv from each clone was analyzed. Thenucleotide sequences thus obtained are summarized in Table 3 and wereconverted into IgG forms. Four antibodies which were respectivelyproduced from clones L3-1, L3-2, L3-3, and L3-5 were used in thesubsequent experiments.

TABLE 3 Library Clone constructed CDR Sequence H11-4 CDR-H1 PEYYMS(SEQ ID NO: 22) YC151 CDR-H1 PDYYMS (SEQ ID NO: 23) YC193 CDR-H1 SDYYMS(SEQ ID NO: 24) YC244 CDR-H2 RNNANGNT (SEQ ID NO: 25) YC321 CDR-H2RNKVNGYT (SEQ ID NO: 26) YC354 CDR-H3 DNWLSY (SEQ ID NO: 27) YC374CDR-H3 DNWLTY (SEQ ID NO: 28) L1-1 CDR-L1 KSSHSLLASGNQNNYLA(SEQ ID NO: 29) L1-3 CDR-L1 KSSRSLLSSGNHKNYLA (SEQ ID NO: 30) L1-4CDR-L1 KSSKSLLASGNQNNYLA (SEQ ID NO: 31) L1-12 CDR-L1 KSSRSLLASGNQNNYLA(SEQ ID NO: 32) L1-22 CDR-L1 KSSHSLLASGNQNNYLA (SEQ ID NO: 33) L2-9CDR-L2 WASKRVS (SEQ ID NO: 34) L2-12 CDR-L2 WGSTRVS (SEQ ID NO: 35)L2-16 CDR-L2 WGSTRVP (SEQ ID NO: 36) L3-1 CDR-L3 QQSYSRPYT(SEQ ID NO: 13) L3-2 CDR-L3 GQSYSRPLT (SEQ ID NO: 14) L3-3 CDR-L3AQSYSHPFS (SEQ ID NO: 15) L3-5 CDR-L3 QQSYSRPFT (SEQ ID NO: 16) L3-32CDR-L3 QQSYSKPFT (SEQ ID NO: 37)

1.7. Conversion of Selected Antibodies into IgG

Respective polynucleotides encoding heavy chains of the four selectedantibodies were designed to have the structure of “EcoRI-signalsequence-VH-NheI-CH-XhoI” (SEQ ID NO: 38). The heavy chains of huAbF46antibodies were used as they were because their amino acids were notchanged during affinity maturation. In the case of the hinge region,however, the U6-HC7 hinge (SEQ ID NO: 57) was employed instead of thehinge of human IgG1. Genes were also designed to have the structure of“EcoRI-signal sequence-VL-BsiWI-CL-XhoI” for the light chain.Polypeptides encoding light chain variable regions of the fourantibodies which were selected after the affinity maturation weresynthesized in Bioneer. Then, a DNA fragment having the heavy chainnucleotide sequence (SEQ ID NO: 38) and DNA fragments having the lightchain nucleotide sequences (DNA fragment including L3-1-derived CDR-L3:SEQ ID NO: 58, DNA fragment including L3-2-derived CDR-L3: SEQ ID NO:59, DNA fragment including L3-3-derived CDR-L3: SEQ ID NO: 60, and DNAfragment including L3-5-derived CDR-L3: SEQ ID NO: 61) were digestedwith EcoRI (NEB, R0101S) and XhoI (NEB, R0146S) before cloning into apOptiVEC™-TOPO TA Cloning Kit enclosed in an OptiCHO™ Antibody ExpressKit (Cat no. 12762-019, Invitrogen) and a pcDNA™3.3-TOPO TA Cloning Kit(Cat no. 8300-01), respectively, so as to construct recombinant vectorsfor expressing affinity-matured antibodies.

Each of the constructed vectors was amplified using Qiagen Maxiprep kit(Cat no. 12662), and a transient expression was performed usingFreestyle™ MAX 293 Expression System (invitrogen). 293 F cells were usedfor the expression and cultured in FreeStyle™ 293 Expression Medium in asuspension culture manner. One day prior to the transient expression,the cells were provided in the concentration of 5×10⁵ cells/ml, andafter 24 hours, when the cell number reached to 1×10⁶ cells/ml, thetransient expression was performed. A transfection was performed by aliposomal reagent method using Freestyle™ MAX reagent (Invitrogen),wherein in a 15 ml tube, the DNA was provided in the mixture ratio of1:1 (heavy chain DNA:light chain DNA) and mixed with 2 ml of OptiPro™SFM (Invitrogen) (A), and in another 15 ml tube, 100 ul (microliter) ofFreestyle™ MAX reagent and 2 ml of OptiPro™ SFM were mixed (B), followedby mixing (A) and (B) and incubating for 15 minutes. The obtainedmixture was slowly mixed with the cells provided one day before thetransient expression. After completing the transfection, the cells wereincubated in 130 rpm incubator for 5 days under the conditions of 37°C., 80% humidity, and 8% CO₂.

After centrifugation, the supernatant was applied to AKTA prime (GEHealthcare) to purify the antibody. In this regard, 100 mL of thesupernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equippedwith a Protein A column (GE healthcare, 17-0405-03), followed by elutionwith an IgG elution buffer (Thermo Scientific, 21004). The buffer wasexchanged with PBS to purify four affinity-matured antibodies(hereinafter referred to as “huAbF46-H4-A1 (L3-1 origin), huAbF46-H4-A2(L3-2 origin), huAbF46-H4-A3 (L3-3 origin), and huAbF46-H4-A5 (L3-5origin),” respectively).

1.8. Construction of Constant Region- and/or Hinge Region-SubstitutedhuAbF46-H4-A1

Among the four antibodies selected in Reference Example 1.7,huAbF46-H4-A1 was found to be the highest in affinity for c-Met and thelowest in Akt phosphorylation and c-Met degradation degree. In theantibody, the hinge region, or the constant region and the hinge region,were substituted.

The antibody huAbF46-H4-A1 (U6-HC7) was composed of a heavy chainincluding the heavy chain variable region of huAbF46-H4-A1, U6-HC7hinge, and the constant region of human IgG1 constant region, and alight chain including the light chain variable region of huAbF46-H4-A1and human kappa constant region. The antibody huAbF46-H4-A1 (IgG2 hinge)was composed of a heavy chain including a heavy chain variable region, ahuman IgG2 hinge region, and a human IgG1 constant region, and a lightchain including the light chain variable region of huAbF46-H4-A1 and ahuman kappa constant region. The antibody huAbF46-H4-A1 (IgG2 Fc) wascomposed of the heavy chain variable region of huAbF46-H4-A1, a humanIgG2 hinge region, and a human IgG2 constant region, and a light chainincluding the light variable region of huAbF46-H4-A1 and a human kappaconstant region. The histidine residue at position 36 on the human kappaconstant region of the light chain was changed to tyrosine in all of thethree antibodies to increase antibody production.

For use in constructing the three antibodies, a polynucleotide (SEQ IDNO: 63) encoding a polypeptide (SEQ ID NO: 62) composed of the heavychain variable region of huAbF46-H4-A1, a U6-HC7 hinge region, and ahuman IgG1 constant region, a polynucleotide (SEQ ID NO: 65) encoding apolypeptide (SEQ ID NO: 64) composed of the heavy chain variable regionof huAbF46-H4-A1, a human IgG2 hinge region, and a human IgG1 region, apolynucleotide (SEQ ID NO: 67) encoding a polypeptide (SEQ ID NO: 66)composed of the heavy chain variable region of huAbF46-H4-A1, a humanIgG2 region, and a human IgG2 constant region, and a polynucleotide (SEQID NO: 69) encoding a polypeptide (SEQ ID NO: 68) composed of the lightchain variable region of huAbF46-H4-A1, with a tyrosine residue insteadof histidine at position 36, and a human kappa constant region weresynthesized in Bioneer. Then, the DNA fragments having heavy chainnucleotide sequences were inserted into a pOptiVEC™-TOPO TA Cloning Kitenclosed in an OptiCHO™ Antibody Express Kit (Cat no. 12762-019,Invitrogen) while DNA fragments having light chain nucleotide sequenceswere inserted into a pcDNA™3.3-TOPO TA Cloning Kit (Cat no. 8300-01) soas to construct vectors for expressing the antibodies.

Each of the constructed vectors was amplified using Qiagen Maxiprep kit(Cat no. 12662), and a transient expression was performed usingFreestyle™ MAX 293 Expression System (invitrogen). 293 F cells were usedfor the expression and cultured in FreeStyle™ 293 Expression Medium in asuspension culture manner. One day prior to the transient expression,the cells were provided in the concentration of 5×10⁵ cells/ml, andafter 24 hours, when the cell number reached to 1×10⁶ cells/ml, thetransient expression was performed. A transfection was performed by aliposomal reagent method using Freestyle™ MAX reagent (Invitrogen),wherein in a 15 ml tube, the DNA was provided in the mixture ratio of1:1 (heavy chain DNA:light chain DNA) and mixed with 2 ml of OptiPro™SFM (Invitrogen) (A), and in another 15 ml tube, 100 ul (microliter) ofFreestyle™ MAX reagent and 2 ml of OptiPro™ SFM were mixed (B), followedby mixing (A) and (B) and incubating for 15 minutes. The obtainedmixture was slowly mixed with the cells provided one day before thetransient expression. After completing the transfection, the cells wereincubated in 130 rpm incubator for 5 days under the conditions of 37°C., 80% humidity, and 8% CO₂.

After centrifugation, the supernatant was applied to AKTA prime (GEHealthcare) to purify the antibody. In this regard, 100 mL of thesupernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equippedwith a Protein A column (GE healthcare, 17-0405-03), followed by elutionwith IgG elution buffer (Thermo Scientific, 21004). The buffer wasexchanged with PBS to finally purify three antibodies (huAbF46-H4-A1(U6-HC7), huAbF46-H4-A1 (IgG2 hinge), and huAbF46-H4-A1 (IgG2 Fc)).Among the three antibodies, huAbF46-H4-A1 (IgG2 Fc) was selected for thefollowing examples, and name as L3-1Y/IgG2 (or SAIT301).

Example 1 Measurement of the Level of PDGF in Anti-c-Met AntibodyResistance Acquired Cell Line

To examine the quantitative change of PDGF when a resistance to ananti-c-Met antibody is induced, SAIT301 resistance acquired EBC1-SR#6cell lines were prepared by repeatedly treating EBC1 (JCRB 0820) cellswith SAIT301. EBC1 parent cells were all responsive to SAIT301, beforeinducing SAIT301 resistance. The SAIT301 resistance acquired EBC1-SR#6cell lines were prepared as follows:

EBC1 (JCRB 0820) cell lines was treated with SAIT301 for at least 2months, with increasing the concentration of treated antibody. Theconcentration of SAIT301 was increased from 1 ug/ml to 10 ug/ml until aresistance is induced. To confirm the acquisition of a resistance toSAIT301, the prepared clones were treated with SAIT301 at theconcentration of 0 ug/ml, 0.016 ug/ml, 0.08 ug/ml, 0.4 ug/ml, or 2ug/ml, and 72 hours after the antibody treatment, the number of theliving cells was counted by CellTiter Glo assay (Promega, G7573). Cloneswhere SAIT301 exhibited no effect were identified.

The obtained SAIT301 resistance acquired cell lines were namedEBC1-SR#6.

The ligand assay was performed using Human Growth Factor Antibody Array(RayBiotech Inc.) to compare the expression of various human growthfactors in EBC1 cell lines and EBC1-SR#6 cell lines with acquiredresistance to an anti-c-Met antibody. In particular, 1×10⁶ of EBC1 andEBC1-SR#6 cells were stabilized for 24 h in 100 mm dish respectively,followed by incubation in a serum-free medium without FBS for 24 h, thenthe medium were collected. One ml of the collected cell medium was addedto a membrane which was protected with buffer, followed by incubation at4° C. for 24 hr and washed. Then 1 ml of biotin-conjugated primaryantibody was added, followed by incubation at 4° C. for 24 hr and washedagain. Two ml of Horseradish peroxidase labeled streptavidin was added,followed by incubation at RT for 1 hr, and exposed on X-ray filmdeveloped to detect signal strength.

The measured amount of the protein in resistance acquired cell linesEBC1-SR#6 was compared to a parent cell EBC1 which is a cell beforeacquisition of resistance, and the results are shown in FIG. 1. As shownin FIG. 1, the PDGF level was significantly increased in EBC1-SR#6 celllines after acquisition of resistance, compared to that beforeacquisition of resistance, whereas the expression level of human growthfactors such as GM-CSF (granulocyte macrophage colony-stimulatingfactor), IGFBP6 (insulin-like growth factor binding protein 6) and VEGF(Vascular endothelial growth factor) was not different regardless ofwhether or not a resistance to anti-cMet antibody was acquired.

Example 2 Identification of Whether an Increase of the Expression Levelof PDGF Contributes to Obtaining Resistance to Anti-cMet Antibody

To examine whether the quantitative increase of PDGF protein contributesto obtaining resistance in anti-c-Met antibody resistance acquired cellline, EBC1 and EBC1-SR#6 cell lines were treated with PDGFR (PDGFReceptor) TKI (tyrosine kinase inhibitor), then the changes of cellgrowth inhibition were measured using EZ-Cytox Cell viability assay kit(DaeiLab Service, Seoul, Korea).

For this, each of EBC1 cells and EBC1-SR#6 cells were seeded on 96-wellplate at the amount of 5000 (5×10³) cells. Twenty four hours after, thecells were solely treated with each of SAIT301, PDGFR inhibitorPonatinib (Selleckchem USA), or FGFR (fibroblast growth factor receptor)inhibitor PD173074 (Selleckchem USA) as Negative control. Seventy twohours after the treatment, the number of the cells was measured byEZ-Cytox Cell viability assay kit.

As shown in FIG. 2, chemosensitivity of EBC1-SR#6 cell line (i.e.resistance acquired cell line) was increased (i.e. IC₅₀ was decreased)than that of EBC1 (i.e. parental cell line), when EBC1 and EBC1-SR#6cell lines were treated with PDGFR inhibitor Ponatinib. In contrast, asshown in FIG. 3, chemosensitivity of EBC1-SR#6 cell line was not changedmarkedly, when EBC1 cell lines and EBC1-SR#6 cell lines were treatedwith FGFR inhibitor PD173074 as Negative control. These finding suggestthat increased expression level of PDGF after the acquisition of theresistance to an anti-c-Met antibody plays an important role in cellgrowth and proliferation of EBC1-SR#6 cell lines.

It should be understood that the exemplary embodiments described hereinshould be considered in a descriptive sense only and not for purposes oflimitation. Descriptions of features or aspects within each embodimentshould typically be considered as available for other similar featuresor aspects in other embodiments.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and “at least one” andsimilar referents in the context of describing the invention (especiallyin the context of the following claims) are to be construed to coverboth the singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The use of the term “at least one”followed by a list of one or more items (for example, “at least one of Aand B”) is to be construed to mean one item selected from the listeditems (A or B) or any combination of two or more of the listed items (Aand B), unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

What is claimed is:
 1. A method for evaluating efficacy of a c-Mettargeting agent in a patient with cancer, or the patient's resistance tothe c-Met targeting agent, the method comprising: measuring PDGF proteinlevel, mRNA expression level of a gene encoding PDGF, or both, in asample obtained from a patient with cancer before administration of ac-Met targeting agent to the patient, and in a sample obtained from thepatient after administration of a c-Met targeting agent to the patient,comparing the PDGF protein level of the samples, mRNA expression levelof gene encoding PDGF, or both, of the samples obtained beforeadministration of the c-Met targeting agent to that of the samplesobtained after administration of the c-Met targeting agent, andevaluating whether the c-Met targeting agent is effective in treatingthe cancer based on a change in PDGF protein level, mRNA expressionlevel of a gene encoding PDGF, or both, wherein an increase in the PDGFprotein level or the mRNA expression level of gene encoding PDGF in thesample from the patient after administration of the c-Met targetingagent, compared to the PDGF protein level or the mRNA expression levelin the sample before administration of the c-Met targeting agent,indicates that the c-Met targeting agent is not efficacious in treatingthe cancer or the patient has resistance to the c-Met targeting agent.2. The method according to claim 1, wherein measuring the mRNAexpression level of gene encoding PDGF comprises contacting the samplewith an oligonucleotide, a primer, or a probe that specifically binds tothe mRNA transcript of the gene encoding PDGF.
 3. The method accordingto claim 1, wherein measuring the PDGF protein level comprisescontacting the sample with an antibody, an antibody fragment or anaptamer that specifically binds to PDGF protein.
 4. The method accordingto claim 1, wherein the c-Met targeting agent comprises an anti-c-Metantibody or an antigen-binding fragment thereof.
 5. The method accordingto claim 4, wherein the anti-c-Met antibody or the antigen-bindingfragment thereof recognizes or binds to a polypeptide comprising 5 to 19contiguous amino acid residues within the SEMA domain (SEQ ID NO: 79) ofc-Met protein and wherein the polypeptide comprises at least the aminosequence of SEQ ID NO: 73
 6. The method according to claim 4, whereinthe anti-c-Met antibody or the antigen-binding fragment thereofcomprises: (i) at least one heavy chain complementarity determiningregion (CDR) selected from the group consisting of (a) a CDR-H1comprising SEQ ID NO: 4; (b) a CDR-H2 comprising SEQ ID NO: 5, SEQ IDNO: 2, or an amino acid sequence comprising 8-19 consecutive amino acidswithin SEQ ID NO: 2 comprising amino acid residues from the 3^(rd) to10^(th) positions of SEQ ID NO: 2; and (c) a CDR-H3 comprising SEQ IDNO: 6, SEQ ID NO: 85, or an amino acid sequence comprising 6-13consecutive amino acids within SEQ ID NO: 85 comprising amino acidresidues from the 1^(st) to 6^(th) positions of SEQ ID NO: 85; (ii) atleast one light chain complementarity determining region (CDR) selectedfrom the group consisting of (a) a CDR-L1 comprising SEQ ID NO: 7, (b) aCDR-L2 comprising SEQ ID NO: 8, and (c) a CDR-L3 comprising SEQ ID NO:9, SEQ ID NO: 15, SEQ ID NO: 86, or an amino acid sequence comprising9-17 consecutive amino acids SEQ ID NO: 89 comprising amino acidresidues from the 1^(st) to 9^(th) positions of SEQ ID NO: 89; or (iii)a combination of the at least one heavy chain complementaritydetermining region and at least one light chain complementaritydetermining region.
 7. The method according to claim 4, wherein theanti-c-Met antibody or the antigen-binding fragment thereof comprises: aheavy chain variable region comprising a polypeptide (CDR-H1) comprisingSEQ ID NO: 1, 22, 23, or 24, a polypeptide (CDR-H2) comprising SEQ IDNO: 2, 25, or 26, and a polypeptide (CDR-H3) comprising SEQ ID NO: 3,27, 28, or 85; and a light chain variable region comprising apolypeptide (CDR-L1) comprising SEQ ID NO: 10, 29, 30, 31, 32, 33 or106, a polypeptide (CDR-L2) comprising SEQ ID NO: 11, 34, 35, or 36, anda polypeptide (CDR-L3) comprising SEQ ID NO: 12, 13, 14, 15, 16, 37, 86,or
 89. 8. The method according to claim 4, wherein the anti-c-Metantibody or the antigen-binding fragment thereof comprises: a heavychain variable region comprising SEQ ID NO: 17, 74, 87, 90, 91, 92, 93,or 94, a light chain variable region comprising SEQ ID NO: 18, 19, 20,21, 75, 88, 95, 96, 97, 98, 99, or 107, or a combination of the heavychain variable region and the light chain variable region.
 9. The methodaccording to claim 4, wherein the anti-c-Met antibody or theantigen-binding fragment thereof comprises: a heavy chain comprising SEQID NO: 62, the amino acid sequence from the 18^(th) to 462^(nd)positions of SEQ ID NO: 62, SEQ ID NO: 64, the amino acid sequence fromthe 18^(th) to 461^(st) positions of SEQ ID NO: 64, SEQ ID NO: 66, orthe amino acid sequence from the 18^(th) to 460^(th) positions of SEQ IDNO: 66; and a light chain comprising SEQ ID NO: 68, the amino acidsequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 68, SEQ IDNO: 70, the amino acid sequence from the 21^(st) to 240^(th) positionsof SEQ ID NO: 70, or SEQ ID NO:
 108. 10. The method according to claim4, wherein the anti-c-Met antibody or the antigen-binding fragmentthereof comprises a heavy chain consisting of the amino acid sequencefrom the 18^(th) to 460^(th) positions of SEQ ID NO: 66, and a lightchain consisting of the amino acid sequence from the 21^(st) to 240^(th)positions of SEQ ID NO: 68.